Composite chromatographic sorbent of mineral oxide beads with hydroxyapatite-filled pores

ABSTRACT

A new adsorbent of a porous mineral oxide material with apatite crystals, preferably hydroxyapatite crystals, in the pores of the mineral oxide material is disclosed. The adsorbent is useful for protein and nucleic acid separations

BACKGROUND OF THE INVENTION

The present invention relates to a new adsorbent of a porous mineraloxide material with apatite crystals, preferably hydroxyapatitecrystals, in the pores of the mineral oxide material. The adsorbent isuseful for protein and nucleic acid separations.

Apatite is a calcium phosphate material in crystalline form having thegeneral formula Ca₅(F, Cl, OH, ½ CO₃) (PO₄)₃. One of the more commontypes of apatite is hydroxyapatite which has the formula[Ca₂(PO₄)₂]₃Ca(OH)₂. It is useful as a packing material to be filled incolumns for chromatographic separation of biopolymers, for example,proteins, enzymes, and nucleic acids. Its ability to adsorb suchmolecules depends on both the structure of the crystal itself and on theexposed surface area of the crystals.

The technique for the preparation of hydroxyapatite utilizable forcolumn chromatography was first developed by Tiselius et al. [Arch.Biochem. Biophys., 65:132-155 (1956)]. Hydroxyapatite for columnchromatographic use has been prepared by various methods.Conventionally, hydroxyapatites are synthesized by (1) wet synthesis inwhich a water-soluble calcium salt and phosphate are allowed to react inaqueous solution, (2) dry synthesis in which calcium phosphate andcalcium carbonate are allowed to react in the presence of water vapor at900° to 1400° C., or (3) hydrothermal synthesis in which calciumhydrogen-phosphate is hydrolyzed, for example, at 200° C. and 15atmospheres. The hydroxyapatites produced in conventional processes havebeen in the form of plates which have to be finely divided, particularlywhen used as column packing material for chromatographic separation. Theplates are divided into tiny pieces varied in shape and size. Theirregular pieces of hydroxyapatite cannot be packed uniformly or denselyin the column for chromatographic separation.

Hydroxyapatite in the form of plate-like crystals or agglomerates ofmicrocrystals also is inferior in mechanical strength and tends to bedestroyed during the packing operation and measurement. Chromatographiccharacteristics of the hydroxyapatite vary according to the packingmethod used, leading to variability in separations and bed collapse.

In recent years, a process for producing microspherical hydroxyapatitewas proposed to overcome these shortcomings, utilizing the so-calledspray-drying method which is widely used for manufacturing granules of apowdery substance (Japanese Laid-open Patent Appln. Nos. Sho. 62-206445and 62-230607). According to the process disclosed in Japanese Laid-openPatent Appln. No. 62-206445, microcrystals of hydroxyapatite having adiameter of less than 1 μm as primary particles are physicallycoagulated by spray drying to form substantially spherical particles of1-10 μm in diameter as second particles.

When the spherical hydroxyapatite particles obtained according to theseprocesses are subjected to classification by screening to collectparticles of a definite particle size as a packing material for liquidchromatography, the spherical particles tend to be destroyed because oftheir poor mechanical strength and are broken to pieces when packeddensely in a column under high pressure. Consequently, the sphericalhydroxyapatite particles formed by spray drying have to be subjected toa heat treatment carried out at a high temperature for a long period oftime in order to impart mechanical strength sufficient enough towithstand high pressure on packing. The severe heat treatment, however,causes the spherical particles tend to be bonded to one another in amutually fused state to form partially solid state granules.

Japanese Laid-open Patent Appin. No. Sho. 62-230607 discloses a processfor preparing spherical agglomerates of apatite in which a gelledhydroxyapatite slurry is sprayed into an atmosphere kept at 100-200° C.to form spherical agglomerates of hydroxyapatite having a diameter of1-10 μm. Hydroxyapatite trapped in a hydrogel network is relativelysoft, and binding capacity is modest because of the limited amount ofhydroxyapatite crystals present in a given volume of sorbent, about 40%.The presence of a hydrogel that surrounds crystals of hydroxyapatiteprevents the direct contact with very large molecules such as plasmids.

Thus, the conventional processes involve a number of problems not onlyin the preparation of spherical hydroxyapatite particles but also in theuse of the particles as a packing material for chromatographic purposes.

Mineral oxide beads for use in chromatography are known, and can havemore strength than hydroxyapatite sorbents. For applications in whichanother substance is introduced into the bead, a pore size larger than500 A is required to allow for unhindered diffusion of large molecules.It is difficult to obtain a large pore diameter, however, withoutadversely affecting porosity and strength. Moreover, mineral oxidesurfaces exhibit various types of interactions with proteins, includingelectrostatic, van der Waals, and Lewis acid-base, that can alter thequality of a separation or even denature a biomolecule.

There is a need for relatively small porous particles which provide theseparation capabilities of apatite yet retain their shape, theirchemical and mechanical properties in specific environments useful forbiomolecule separation in columns as well as in suspensions, and whichoffer a substantial density difference with liquids used in adsorptionand chromatography. Such apatite materials excellent in mechanicalstrength and chromatographic characteristics have not, as yet, beendescribed.

SUMMARY OF THE INVENTION

In accordance with the present invention, a chromatography sorbent isprovided which combines the separation capabilities of hydroxyapatitecrystals with the strength of a mineral oxide network. This compositesorbent exhibits superior properties when used in chromatographicseparation, or in substance separation or development when used as theadsorbent packed or charged into a column or as a stationary phase agentin column chromatography. Chromatography using this sorbent as theadsorbent in batch separations or packed in either a fixed bed orfluidized bed column achieves high acuteness and precision separationand fractionation of substances having a minute difference in structurefrom one another. This was difficult to achieve with the use ofprior-art adsorbents. Such substances may include biologicalmacromolecule materials having a molecular weight of 10⁴ to 10⁹ Dalton,such as proteins, including immunoglobulins, interferon or enzymes, ornucleic acids, such as RNA, DNA or plasmids or viruses. The compositesorbent is indispensable for high purity separation and refining of avariety of ultimately useful substances obtained by gene recombination,cell fusion or cell culture en masse.

The composite chromatographic sorbent comprises mineral oxide beads withpores filled with apatite, particularly hydroxyapatite. The mineraloxide beads of the composite sorbent are characterized by high porosity,low surface area, high mean pore diameter, and high mechanicalstability. Moreover, they show a density that facilitates packing offixed-bed columns, increases the particle sedimentation velocity inbatch, and permits the use of high velocity in fluidized-bed operations.The apatite crystals are protected by a very strong skeleton of mineraloxide, preferably zirconia.

Specifically, the present invention encompasses a compositechromatography sorbent comprising porous mineral oxide beads that have apore volume which exceeds about 10% of the bead volume, preferably about30% and about 70%, and more preferably about 30% and about 60%, and anaverage pore diameter of at least about 500 Å, preferably between about1000 Å and about 4000 Å, and more preferably between about 1000 Å andabout 3000 Å. The pores of the beads contain apatite, and preferablyhydroxyapatite, crystals. Preferably, the mineral oxide is selected fromthe group consisting of alumina, titania, hafnia, silica, zirconia andmixtures thereof, and most preferably it is zirconia. In a preferredembodiment, the mineral is silica, the pore volume is between about 40%and about 70% of the bead volume, and the average pore diameter isbetween about 2000 Å and about 5000 Å.

In one embodiment, the beads of the composite chromatography sorbent arecoated with a layer of hydrophilic polymer, preferably a hydrophilicpolymer selected from the group consisting of polyoxyethylene,polyoxypropylene, cross-linked polysaccharides and vinyl polymers.

In a preferred embodiment, the apatite crystals comprise calcium ionsand a metal ion or a metalloid ion. Preferably, the metal ion ormetalloid ion is strontium, barium or fluoride.

The present invention also encompasses chromatography apparatus andmethods. A chromatography column comprises a tubular member having aninlet end and an outlet end; first and second porous members disposedwithin the tubular member; and a composite chromatography sorbentaccording to the invention packed within the tubular member between thefirst and second porous members. Preferably, the column volume isbetween about 50 liters and about 5000 liters. The column additionallymay comprise means for flowing a liquid sample upward through thecomposite chromatography sorbent.

The column may be used in a chromatographic separation method comprisingflowing a solution comprising biomolecules through the chromatographycolumn so that the solution permeates the pores of the mineral oxidebeads, wherein some biomolecules in the solution are bound to theapatite crystals and other, different biomolecules remain in thesolution. This may be followed by flowing another solution through thechromatography column to elute the biomolecules bound to the apatitecrystals. In one embodiment the biomolecules are polypeptides, and inanother embodiment the biomolecules are nucleic acids. Substances otherthan proteins and nucleic acids are included within the scope of theterm biomolecule, such as glycopeptides.

The composite sorbent may also be used in batch chromatography apparatusand methods. In a batch method, a solution comprising biomolecules isbrought into contact with the composite chromatography sorbent, so thatthe solution permeates the pores of the mineral oxide beads, whereinsome biomolecules in the solution are bound to the apatite crystals andother, different biomolecules remain in the solution.

The present invention also provides a method of making a compositechromatography sorbent according to the invention, comprising: (1)providing porous mineral oxide beads that have a pore volume whichexceeds about 10% of the bead volume and an average pore diameter of atleast about 500 Å; (2) contacting the porous mineral oxide beads with amaximum of one pore volume of a solution of either (i) calcium chlorideor (ii) potassium or sodium phosphate so that it permeates the pores ofthe beads; (3) drying the beads; (4) contacting the dried beads with amaximum of one pore volume of a solution of the other of either (i)calcium chloride or (i) potassium or sodium phosphate so that itpermeates the pores of the beads, thereby forming calcium phosphate inthe pores; (5) washing the beads with water to eliminate excess calciumor phosphate ions; (6) contacting the washed beads with a solution ofsodium hydroxide; (7) washing the beads with water; and (8) contactingthe washed beads with a solution of disodium phosphate to formhydroxyapatite crystals in the pores of the beads. In a preferredembodiment, the beads are washed with a phosphoric acid solution beforecontacting the porous mineral oxide beads with a maximum of one porevolume of a solution of either (i) calcium chloride or (ii) potassium orsodium phosphate.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory and are intended to providefurther explanation of the invention as claimed. Other objects,advantages, and novel features will be readily apparent to those skilledin the art from the following detailed description of the invention.

Detailed Description of Preferred Embodiments

The composite chromatography sorbent according to the invention usesmineral oxide beads to impart mechanical stability to an apatitesorbent. The mineral oxide beads have a pore volume of at least about10% of the bead volume and an average pore diameter of at least about500 Å.

In a preferred embodiment, mineral oxide beads with a higher porevolume, preferably at least about 30%, more preferably at least about40%, and most preferably at least about 50%, are used. The preparationof mineral oxide beads having high pore volumes is described in WO99/51335, the contents of which are incorporated herein in theirentirety. The beads can be small discrete beaded particles as well asirregular shaped particles, showing high pore volume and high mechanicaland chemical stability. Because of their stability and high porosity,they are particularly useful in packed bed, fluidized bed or stirredbatch adsorption or chromatographic separation for large macromolecules.

The mineral oxide beads are prepared by combining a tetravalent metaloxide with a trivalent metal salt or oxide as a pore inducing agent. Thecombination results in the formation of unstable suspensions which,after agglomeration to form spherical or irregular particles, show bothmacroporosity and large pore sizes. The porosity and pore size isgreater than that which can be obtained in the absence of the trivalentmetal salt or oxide.

The mineral oxide preferably is an oxide of titania, zirconia, silica orhafnium, preferably silica or zirconia, and most preferably zirconia.The mineral oxide can also be a mixture of two or more tetravalent metaloxides. Preferably the mineral oxide powders are in the form of apowder, and most preferably a powder with a particle size of about 0.1to about 10 μm.

The trivalent metal can be used in the form of an oxide, a salt, ormixtures of oxide and salt. A particularly preferred salt is nitrate.The metal can be any metal which exhibits a +3 valence, such as GroupIIIB metals, rare earth metals, and the like. Preferred trivalent metalsare aluminum, gallium, indium, scandium, yttrium, lanthanum, cerium,neodymium, erbium, ytterbium and actinium. Also included arecompositions in which the trivalent metal oxide or salt is a mixture oftwo or more such oxides or salts. Such mixtures include salt/oxide,salt/salt, and oxide/oxide mixtures of the same or different trivalentmetals.

Only a limited pore volume reduction is observed when firing thecompositions at very high temperatures. In contrast, mineral oxide beadsobtained without the use of the trivalent metal salt or oxide have lowerpore volumes when fired at very high temperatures, due to a severereduction of pore volume resulting from the firing process. Thetrivalent metal salt or oxide additionally stabilizes a crystalline formof the mineral oxide and prevents grain growth and cracking of the finalmaterial.

Optionally, an agent that induces particle agglomeration to make abeaded final material, such as an agglomeration promoting material or abinder, may be included. These may be salts of trivalent or tetravalentmetals, and can contain the same tetravalent or trivalent metals justdescribed. In a preferred embodiment, the binder comprises a mixture ofnitrates, including a tetravalent metal nitrate and trivalent metalnitrate. For example, when zirconium oxide is used as a mineral oxidebead constituent and cerium oxide is used as the trivalent pore inducingagent, it is convenient to use a mixture of zirconium nitrate and ceriumnitrate as a binder. Other suitable binders include materials which formmineral hydrogels that can encapsulate mineral oxide elementalparticles, for example, silica gels. A mineral hydrogel may also be usedin combination with one or more additional binders.

Composite mineral oxides with enhanced pore volume are made by preparinga liquid suspension of a tetravalent mineral oxide. The liquid portionof the suspension can be water or any other appropriate solvent. Themineral oxide should be in the form of a powder, with a particle size ofbetween about 0.1 and about 10 μm, with the particular particle sizechosen depending on the desired pore size of the porous particles. Thissuspension is mixed with one or more pore inducing trivalent agents. Thesuspension optionally also contains one or more binders.

In a typical composition which includes one or more metal oxide or saltbinders, the binders are first mixed in a liquid such as water, then themineral oxide and the pore inducing agent are added while stirring,producing a suspension. The stirring should be gentle to avoidintroducing air bubbles into the mixture.

The amount of pore inducing agent which is used in the initialsuspension is roughly proportional to the amount of mineral oxide used.In the final product, the oxide of the tetravalent metal will constituteabout 50 to about 99% of the final particles, with the remaining about 1to about 50% made up of pore inducers and optional binders. In theinitial suspension, however, the mineral oxide particles, the majorconstituent of the porous beads, are at a concentration of about 10 toabout 95% by weight, based on the total weight of components used. Morepreferably, the mineral oxide should be about 20 to about 60% by weight.The pore inducing agent concentration is between about 5 and about 50%by weight. The optimal concentration varies, depending on the nature ofthe specific compounds used. The concentration of the agglomerationpromoting material or binder is between about 0 and about 20% by weight,and also depends on the nature of the binders. Optionally, organiccompounds may also be added to the initial suspension in order to alterthe viscosity of the solution.

The suspension containing all of the desired components is then used toform beads. A variety of techniques well known in the art, such as spraydrying, emulsion-polycondensation and sol-gel processes can be used toeffect the agglomeration on the compositions. After the elementalparticles are agglomerated into a beaded shape, they are heated at hightemperatures to stabilize the architecture of the porous mineral bead bypartial fusion of the elemental particles. The heating rate, thecalcination temperature and the soak time used depend on the nature ofthe mineral oxides and mineral pore inducers. A controlled sintering isdesirable in order to obtain stronger particles without elimination ofthe porosity. Typically, temperatures between about 800 and about 1400°C., for a duration of about 1 to about 10 hours, and with a heating rateranging between about 1 and about 100° C./hour, are used. A sequentialcalcination treatment also can be used, to first remove volatilecomponents such as water, organic materials, nitrates and the like, andthen to sinter the elemental particles.

The fired beads then are cooled to room temperature, and subsequentlywashed with, for example, acidic, alkaline, neutral or dilutedhydro-organic solutions. The particles optionally can be subjected to asieving step to adjust the particle size distribution, as desired.Typical pore volumes of at least about 30%, about 40% or about 50% canbe obtained according to the invention. The upper limit of pore volumeis about 70%.

The beads with larger pore volumes and/or average pore diameters areparticularly suited for the introduction of apatite crystals, preferablyhydroxyapatite, to prepare a composite chromatography sorbent. Porevolume varies based on the bead material. For example, when the mineraloxide is selected from the group consisting of zirconia, titania andhafnia, the pore volume is between about 30% and about 60% of the beadvolume. When the mineral is silica and the pore volume is between about40% and about 70% of the bead volume.

Pore size also varies depending on the bead material, and can beselected based on the material to be separated by the composite sorbent.Larger average pore diameter is selected for applications in separatinglarger biomolecules, such as plasmids, in which an average pore diametergreater than 2000 Å may be required. The average pore diameter generallyis between about 1000 Å and about 4000 Å. When the mineral is selectedfrom the group consisting of zirconia, titania and hafnia, the averagepore diameter is between about 1000 Å and about 3000 Å, whereas when themineral is silica the average pore diameter is between about 2000 Å andabout 5000 Å.

In order to fill the pores with hydroxyapatite, mineral oxide beadsoptionally, but preferably, first are washed with a solution ofphosphoric acid to eliminate impurities and then incubated withmono-potassium phosphate. The beads are then washed and dried. Ifdesired, the pore volume can be determined by known methods. The beadsare next contacted with a solution of either (i) calcium chloride or(ii) potassium or sodium phosphate, and the solution is allowed topenetrate the pores. The beads are dried and then contacted with asolution of either (i) calcium chloride or (ii) potassium or sodiumphosphate. If the beads were contacted with calcium chloride in thefirst step, then they are contacted with potassium or sodium phosphatein the second step, and vice versa. The solution is allowed to penetratethe pores, and after allowing sufficient time for the calcium phosphatecrystalline structure to form within the pores, the beads are washed toeliminate excess calcium or phosphate ions. The beads then are contactedwith a solution of sodium hydroxide, and are again washed. Finally, thebeads are contacted with a solution of disodium phosphate to formhydroxyapatite crystals in the pores of the beads.

When the mineral beads are contacted with the calcium chloride andpotassium or sodium phosphate solutions, it is preferable to use amaximum of one pore volume of solution, and preferably exactly one porevolume. The use of one pore volume exactly generates the maximum amountof hydroxyapatite crystals in the pores of the mineral oxide beads,without having crystals grow outside the pores of the beads.

In an alternative embodiment, the hydroxyapatite made with phosphateions can be doped with small amounts of other metal ions. The dopedmetal ions can used to vary the adsorption properties of the compositesorbent.

Apatites other than hydroxyapatite can be grown in the pores. In thiscase, calcium could be replaced by strontium, barium or other elements.The resulting apatites would have different adsorption properties thanhydroxyapatite. Crystalline apatites other than hydroxyapatite, such asapatite derivatives with F, Cl or CO₃, are known can be grown in thepores of the mineral oxide beads. For example, the preparation offluorapatite is described in Matsumoto et al., Caries Res., 34(1):26-32(2000); Okazaki et al., Biomaterials, 20(15):1421-6 (1999); Okazaki etal., Biomaterials, 19(10):919-23 (1998); Okazaki et al., Biomaterials,19(7-9):611-6 (April;-May, 1998).

The apatite crystals, and more preferably the hydroxyapatite crystals,comprise calcium ions and a metal ion or a metalloid ion. In embodimentsusing a metal or metalloid ion, preferred metal ions or metalloid ionsis strontium, barium or fluoride.

Prior to the formation of apatite crystals in the pores of the mineraloxide beads, the beads may first be coated with a layer of hydrophilicpolymer. Preferably, the hydrophilic polymer is selected from the groupconsisting of polyoxyethylene, polyoxypropylene, cross-linkedpolysaccharides and vinyl polymers. The coating reduces non-specificbinding for biomolecules.

Different chromatography techniques can be used to separate biomoleculesusing the composite sorbent according to the invention. These techniquescomprise contacting a solution containing the biological macromoleculeswith the composite sorbent leading to the selective adsorption ormolecules in the solution by the sorbent. In the event of the desiredmacromolecule(s) being fixed to the resin, the elution of the latterallows it or them to be separated and collected in a purified andconcentrated form. If the desired macromolecule remains in the treatedsolution (the other macromolecules being fixed to the sorbent) then thedesired separation is obtained directly.

When using batch chromatography, the composite sorbent is added directlyto the solution of biomolecules, and the sorbent-biomolecule mixture isgently agitated for a time sufficient to allow the biomolecules to bindto the sorbent. The biomolecule-bound-sorbent may then be removed bycentrifugation or filtration, and the biomolecules subsequently elutedin a separate step.

Alternatively, column chromatography may be used. In fixed bed columnchromatography, the composite sorbent is packed into a column, and thesolution which contains the biomolecules to be separated is applied tothe sorbent by pouring it through the sorbent at a rate that allows thebiomolecules to bind to the sorbent. Advantages of fixed bedchromatography include minimal column volume and water consumption. Thedisadvantage of the column chromatography method is that the flow rateof liquids through the column is slow, and, therefore, time-consuming.This flow rate can be reduced even further if the material being appliedto the column includes particulates, since such particulate material can“clog” the sorbent to some degree.

In fluidized bed column chromatography, a rising filtration flow andlarge rather than dense particles are used in order to maintain anequilibrium against the rising forces. An essentially vertical columncomposed of between 2 and 5 stages placed on top of the other is used,and the solution successively passes through stage and is drawn off byan overflow on the upper part of the upper stage. Each stage, with theexception of the uppermost one, is separated by two perforateddistribution systems, one distributing the solution at the base of thestage in question, the other distributing the solution towards the stagelocated immediately above.

The advantages of a fluidized bed are higher flow rates at lowerpressures as compared to fixed bed chromatography. Although the higherflow rates offer certain advantages to the chromatographic separation,the method has several shortcomings. The method requires larger diameterresins that are neutral to gravity or buoyant. These larger diametersorbents have less surface area per unit volume than smaller sorbentsused in fixed bed columns, and correspondingly have less surface bindingcapacity. The most significant problem of the fluidized bed is mixing.Since the column does not contain any static mixing means, the bed isconventionally mixed by means of air jets or by recycling the liquid tobe separated through the column at a high flow rate. The high flow rateand limited mixing inhibit the uniform phase change required duringelution of the product from the resin.

On the other hand, fluidized bed chromatography avoids many of theserious disadvantages of fixed beds, which include clogging, need forcleaning, compression and cleaning-induced resin deterioration. In fact,the fluidized bed allows free passage of impurities in the solution withno risk of clogging; no cleaning is necessary so the life-span of theresins is greatly increased. However, the chromatographic sorbents forbiological macromolecules typically are not suitable for fluidized bedchromatography, being too small in granulometry, or having a density tooclose to that of water. This makes it impossible to fluidize withoutdrawing particles into the flux. Another problem with fluidized bedchromatography of biological macromolecules relates to the large spacebetween molecules, would result in a decrease in efficiency in afluidized bed environment.

Based on these factors, batch and fixed bed chromatography have been themethods of choice in prior art separation techniques for biologicalmacromolecules. The present composite sorbent, on the other hand, can beused in a batch, fixed bed, or fluidized bed chromatography.

The composite sorbent according to the invention is used to separatebiomolecules contained in a “source liquid,” which is a liquidcontaining at least one and possibly two or more biological substancesor products of value which are sought to be purified from othersubstances also present. In the practice of the invention, sourceliquids may for example be aqueous solutions, organic solvent systems,or aqueous/organic solvent mixtures or solutions. The source liquids areoften complex mixtures or solutions containing many biological moleculessuch as proteins, antibodies, hormones, and viruses as well as smallmolecules such as salts, sugars, lipids, etc. While a typical sourceliquid of biological origin may begin as an aqueous solution orsuspension, it may also contain organic solvents used in earlierseparation steps such as solvent precipitations, extractions, and thelike. Examples of source liquids that may contain valuable biologicalsubstances amenable to the purification method of the invention include,but are not limited to, a culture supernatant from a bioreactor, ahomogenized cell suspension, plasma, plasma fractions, milk, colostrumand cheese whey.

The source liquid contains at least one “biomolecule” to be purifiedfrom the source liquid. Biomolecules are biological products andinclude, for example, nucleic acids, immunoglobulins, clotting factors,vaccines, antigens, antibodies, selected proteins or glycoproteins,peptides, enzymes, etc. The biomolecule may be present in the sourceliquid as a suspension or in solution. For convenience, the term“biomolecule” is used herein in the singular, but it should beunderstood that it may refer to more than one substance that is to bepurified, either together as co-products or separately (e.g.,sequentially) as discrete recovered components.

An “elution liquid” or “elution buffer” is used to dissociate thebiomolecules, such as glyco-iso-forms, away from the composite sorbent.The elution liquid acts to dissociate the biomolecules withoutdenaturing them irreversibly. Typical elution liquids are well known inthe chromatography art and may have higher concentrations of salts, freeaffinity ligands or analogs, or other substances that promotedissociation of the target substance from the chromatography sorbent.“Elution conditions” refers to process conditions imposed on thebiomolecule-bound chromatography sorbent that dissociate the undenaturedbiomolecules from the chromatography sorbent, such as the contacting ofthe biomolecule-bound chromatography sorbent with an elution liquid orelution buffer to produce such dissociation.

A “cleaning liquid” or “cleaning buffer” is used to wash thechromatography sorbent after the completion of the separation process.The cleaning liquid may contain a detergent, a virus-inactivating agent,or relatively high concentrations of salts, and may have a higher orlower pH than the liquids used during the purification process. Itspurpose is to fully decontaminate the chromatography sorbent to renderit ready for reuse. Typical cleaning liquids are well-known in thechromatography art.

Between uses, the composite sorbent is stored in a “storage liquid” or“storage buffer.” Storage liquids, in addition to buffering ions, mayalso contain microbicides or other preservatives. Such storage liquidsare well known in the chromatography art.

The composite sorbent can be used in batch separations, or it can bepacked into a chromatography column, either a fixed bed or fluidizedbed. The column comprises a tubular member having an inlet end and anoutlet end, and first and second porous members, such as a glass frit,disposed within the tubular member. The composite chromatography sorbentis packed within the tubular member between the first and second porousmembers. In a fluidized bed column, there typically are multiple stages.In a preferred embodiment, the column volume is between about 50 litersand about 5000 liters. For fluidized bed chromatography, the columnadditionally comprises a means for flowing a liquid sample upwardthrough the composite chromatography sorbent.

Batch chromatographic separations comprised mixing the composite sorbentwith the source liquid in a suitable container, and gently stirring.Chromatographic separations using column chromatography comprise thesteps of flowing a first solution comprising biomolecules through thecolumn such that the biomolecules in the solution permeate the pores ofthe mineral oxide beads and are bound to the apatite crystals therein;and then flowing a second solution through the column to elute thebiomolecules bound to the apatite crystals. In a fixed bed column thesource liquid flows downwardly by gravity, while in a fluidized bedcolumn the source liquid is propelled upwardly through the column. Thebiomolecules to be separated usually are polypeptides or nucleic acids.The composite sorbent is particularly useful for difficult proteinseparations, including antibody separation. It also is excellent forplasmids for the elimination of RNA and of open circles.

The following examples relate to specific embodiments within the scopeof the present invention, but are not limiting.

EXAMPLE 1 Preparation of Zirconia Particles by Sol-Gel

A silica sol is prepared by mixing sequentially and progressively 150 mlof sodium silicate 35% with 200 ml of water and 100 ml of water and 100ml of glacial acetic acid. Dry solid irregular zirconia powder (350 mgof 0.3 to 3 μm size) is dispersed in this suspension. Cerium oxide (10g) and cerium nitrate (10 g) are then added under vigorous stirring.Under the above conditions the gelation process occurs at ambienttemperature within 15 to 60 minutes.

After complete gelation, which takes a few hours, the gel is dividedinto small pieces by press-filtering it through a 200 μ sieve. Theparticles are suspended in clear water and recovered by filtration,washed and then dried at 80° C. under an air stream.

The silica gel that entraps the solid zirconia and ceria compositemicroparticles is progressively dehydrated. At this point, the particlesare soft and show only very modest porosity. Then, the particles arefired at 1300° C. for 2 hours. Under these conditions, the silica gel istotally dehydrated and shrinks to such an extent that it forms acontinuous layer around the solid sub-particles. The void betweensubparticles constitutes the macroporosity.

After this treatment, the final pore volume represents more than 50% ofthe whole porous particle volume. The density of the dry irregularparticles is about 2.1 g/cm³. After cooling, the beads do not show anycracks due to volume variation of mineral crystalline forms.

EXAMPLE 2 Preparation of Zirconia Particles by Suspension Polymerization

A silica sol is prepared by mixing sequentially and progressively 150 mlof sodium silicate 35% with 200 ml of water and 100 ml of water and 100ml of glacial acetic acid. Dry solid irregular zirconia powder (350 mgof 0.3 to 3 μm size) is dispersed in this suspension. Cerium oxide (10g) and cerium nitrate (10 g) are then added under vigorous stirring.

The resulting homogeneous suspension is slowly poured in an agitatedparaffin oil bath containing 2% sorbitan sesquioleate and dispersed assmall droplets. The suspension is heated at 80° C. while stirring. Underthese conditions, the gelation process occurs at ambient temperaturewithin 15 to 30 minutes.

The beads of a diameter ranging from 10 to 500 μm comprise a silicahydrogel trapped within its network solid microparticles of pre-formedzirconia and ceria. They are recovered by filtration, washed, and driedat 80° C. under an air stream. The gel is progressively dehydrated andacts as a binder for solid zirconia and ceria composite microparticles.The beads are then fired at 1300° C. for 2 hours, to singer beadsub-particles with minimal pore volume reduction. After this treatment,the final pore volume represents more than 50% of the total bead volume.The density of the dry beads is about 2.1 g/cm³. After cooling, thebeads do not show any cracks due to volume variation of mineralcrystalline forms.

EXAMPLE 3 Preparation of Zirconia Beads by Spray Drying

A solution is prepared by mixing 231 g of zirconium nitrate and 143.6 gof yttrium nitrate in 1000 ml of distilled water. Yttrium oxide (144 g)and zirconia powder (752 g of 0.3 to 3 μm size) are added under gentlestirring to prevent the introduction of air bubbles.

The suspension is then injected into a vertical drying chamber throughan atomization device, such as a revolving disk, a spray nozzle, or anultrasonic nebulizer, together with a hot gas stream, preferably air ornitrogen. The hot gas stream causes rapid evaporation of water from themicrodroplets. The gas is typically injected at 300-350° C. and exitsthe dryer at a temperature slightly above 100° C. Microparticles oforiginal mineral oxides are consolidated into individual aggregates ofspherical shape. Dry microbeads are then fired at a temperature close tothe melting temperature of the zirconium oxide to irreversiblyconsolidate the network. After cooling, the beads do not show any cracksdue to volume variation of mineral crystalline forms. This operationresults in the formation of stable beads with a large pore volume thatexceeds 50% of the bead volume.

EXAMPLE 4 Preparation of Hydroxyapatite Filled Zirconia Beads

The beads from Example 3 are washed with a 1 M solution of phosphoricacid to eliminate impurities and then incubated with two volumes of 1 Mmonopotassium phosphate overnight at room temperature under occasionalshaking. The treated beads are then washed with water until neutral pHand dried under vacuum at 60-80° C. to eliminate all residual water. Thepore volume of the dry beads is determined according to well-knownmethods.

A solution of calcium chloride is prepared by solubilizing 74 g ofCaCl₂.2H₂O in 500 ml of distilled water (final volume). The dry beads oftreated zirconia are mixed with one pore volume of the solution ofcalcium chloride. After 30-60 minutes mixing, to ensure a goodpenetration of the solution into the pores of the mineral material, thebeads are dried again, as above.

A solution of disodium phosphate is prepared by solubilizing 180 g ofNa₂HPO₄.12H₂O in 500 ml water (final volume). The dried beads are mixedwith one pore volume of the solution, and the mixture again isthoroughly shaken for 30-60 minutes to ensure good penetration. Thetemperature is kept at 25-40° C.;, and the material is left overnight.

The material then is mixed with a large volume of water and washedseveral times with water until elimination of the excess calcium ions(no precipitation of Ca(OH)₂ with NaOH should occur). The washedmaterial is added to several volumes (at least 10) of sodium hydroxideat a concentration of 0.5M. The suspension is then brought to 95-100° C.for one hour and left overnight, during which time the temperaturedecreases to room temperature.

The treated material is again extensively washed with water and mixedwith a solution of 3 g/l of disodium phosphate. The pH is adjusted to6.8 and the suspension heated to 95-100° C. for about 20 minutes. Theresulting material is finally washed with water and stored in aphosphate buffer at neutral pH containing 1 M sodium chloride and 20%ethanol.

The present invention provides a novel composite adsorbent, methods ofuse and manufacture. While specific examples have been provided, theabove description is illustrative and not restrictive. Any one or moreof the features of the previously described embodiments can be combinedin any manner with one or more features of any other embodiments in thepresent invention. Furthermore, many variations of the invention will bebecome apparent to those skilled in the art upon review of thespecification. The scope of the invention should, therefore, bedetermined not with reference to the above description, but insteadshould be determined with reference to the appended claims along withthe their full scope of equivalents.

All publication and patent documents cited in this application areincorporated by reference in their entirety for all purposed to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their “invention.”

1-12. (canceled)
 13. A chromatography column, comprising: (a) a tubularmember having an inlet end and an outlet end; (b) first and secondporous members disposed within said tubular member; and (c) a compositechromatography sorbent comprising: porous mineral oxide beads that havea pore volume which exceeds about 10% of the bead volume and an averagepore diameter of at least about 500 Å, wherein the pores of the beadscontain apatite crystals which have been formed within the pores of thebeads by solutions that are allowed to penetrate the pores, and whereinsaid composite chromatography sorbent is packed within said tubularmember between said first and second porous members.
 14. Thechromatography column of claim 13, wherein the column volume is betweenabout 50 liters and about 5000 liters.
 15. The chromatography column ofclaim 13, further comprising: means for flowing a liquid sample upwardthrough said composite chromatography sorbent.
 16. The chromatographycolumn of claim 15, further comprising: a series of stages between saidinlet end and said outlet end.
 17. A chromatographic separation methodcomprising: contacting a solution comprising biomolecules with acomposite chromatography sorbent, wherein said composite chromatographysorbent comprises porous mineral oxide beads that have a pore volumewhich exceeds about 10% of the bead volume and an average pore diameterof at least about 500 Å, wherein the pores of the beads contain apatitecrystals, wherein the solution permeates the pores of the mineral oxidebeads, and wherein some biomolecules in the solution are bound to theapatite crystals and other, different biomolecules remain in thesolution.
 18. A chromatographic separation method comprising: a firstflowing solution comprising biomolecules through a chromatography columncomprising: (a) a tubular member having an inlet end and an outlet end;(b) first and second porous members disposed within said tubular member;and (c) a composite chromatography sorbent comprising porous mineraloxide beads that have a pore volume which exceeds about 10% of the beadvolume and an average pore diameter of at least about 500 Å, wherein thepores of the beads contain apatite crystals, and wherein said compositechromatography is packed within said tubular member between said firstand second porous members, wherein the solution permeates the pores ofthe mineral oxide beads, and wherein some biomolecules in the solutionare bound to the apatite crystals and other, different biomoleculesremain in the solution.
 19. The method of claim 18, further comprising:a second flowing solution through the chromatography column to elute thebiomolecules bound to the apatite crystals.
 20. The method of claim 18,wherein the biomolecules are selected from the group consisting ofpolypeptides, nucleic acids, antibodies, and glyco-iso-forms.
 21. Themethod of claim 18, wherein the composite chromatography sorbent isformed by a method comprising: (i) providing porous mineral oxide beadsthat have a pore volume which exceeds about 10% of the bead volume andan average pore diameter of at least about 500 Å; (ii) contacting theporous mineral oxide beads with a maximum of one pore volume of asolution of either (A) calcium chloride or (B) potassium or sodiumphosphate so that it permeates the pores of the beads; (iii) drying thebeads from (ii); (iv) contacting the dried beads with a maximum of onepore volume of a solution of the other of either (A) calcium chloride or(B) potassium or sodium phosphate so that it permeates the pores of thebeads, thereby forming calcium phosphate in the pores; (v) washing thebeads from (iv) with water to eliminate excess calcium or phosphateions; (vi) contacting the washed beads from (v) with a solution ofsodium hydroxide; (vii) washing the beads from (vi) with water; and(viii) contacting the washed beads from (vii) with a solution ofdisodium phosphate to form hydroxyapatite crystals in the pores of thebeads.
 22. The method according to claim 21, wherein the beads arewashed with a phosphoric acid solution before (ii).
 23. Achromatographic separation method comprising: a first flowing solutioncomprising biomolecules through a chromatography column comprising: (a)a tubular member having an inlet end and an outlet end; (b) first andsecond porous members disposed within said tubular member; (c) acomposite chromatography sorbent comprising porous mineral oxide beadsthat have a pore volume which exceeds about 10% of the bead volume andan average pore diameter of at least about 500 Å, wherein the pores ofthe beads contain apatite crystals, and wherein said compositechromatography is packed within said tubular member between said firstand second porous members; and (d) means for flowing a liquid sampleupward through said composite chromatography sorbent, wherein thesolution permeates the pores of the mineral oxide beads, and whereinsome biomolecules in the solution are bound to the apatite crystals andother, different biomolecules remain in the solution.
 24. The method ofclaim 23, further comprising: a second flowing solution through thechromatography column to elute the biomolecules bound to the apatitecrystals.
 25. The method of claim 23, wherein the biomolecules areselected from the group consisting of polypeptides, nucleic acids,antibodies, and glyco-iso-forms.
 26. The method of claim 23, wherein thecomposite chromatography sorbent is formed by a method comprising: (i)providing porous mineral oxide beads that have a pore volume whichexceeds about 10% of the bead volume and an average pore diameter of atleast about 500 Å; (ii) contacting the porous mineral oxide beads with amaximum of one pore volume of a solution of either (A) calcium chlorideor (B) potassium or sodium phosphate so that it permeates the pores ofthe beads; (iii) drying the beads from (ii); (iv) contacting the driedbeads with a maximum of one pore volume of a solution of the other ofeither (A) calcium chloride or (B) potassium or sodium phosphate so thatit permeates the pores of the beads, thereby forming calcium phosphatein the pores; (v) washing the beads from (iv) with water to eliminateexcess calcium or phosphate ions; (vi) contacting the washed beads from(v) with a solution of sodium hydroxide; (vii) washing the beads from(vi) with water; and (viii) contacting the washed beads from (vii) witha solution of disodium phosphate to form hydroxyapatite crystals in thepores of the beads.
 27. The method according to claim 26, wherein thebeads are washed with a phosphoric acid solution before (ii). 28-29.(canceled)
 30. The chromatography column according to claim 13, whereinthe apatite crystals are hydroxyapatite crystals.
 31. The chromatographycolumn according to claim 13, wherein the mineral oxide is zirconia. 32.The chromatography column according to claim 13, wherein the apatitecrystals are hydroxyapatite crystals and the mineral oxide is zirconia.33. The separation method of claim 17, wherein the apatite crystals arehydroxyapatite crystals.
 34. The separation method of claim 17, whereinmineral oxide is zirconia.
 35. The separation method of claim 17,wherein the apatite crystals are hydroxyapatite crystals and the mineraloxide is zirconia.
 36. The method of claim 18, wherein the apatitecrystals are hydroxyapatite crystals.
 37. The method of claim 18,wherein mineral oxide is zirconia.
 38. The method of claim 18, whereinthe apatite crystals are hydroxyapatite crystals and the mineral oxideis zirconia.
 39. The method of claim 23, wherein the apatite crystalsare hydroxyapatite crystals.
 40. The method of claim 23, wherein themineral oxide is zirconia.
 41. The method of claim 23, wherein theapatite crystals are hydroxyapatite crystals and the mineral oxide iszirconia.
 42. The chromatography column of claim 13, wherein the mineraloxide is selected from the group consisting of alumina, titania, hafnia,silica, zirconia and mixtures thereof.
 43. The chromatography column ofclaim 13, wherein the mineral oxide comprises zirconia.
 44. Thechromatography column of claim 13, wherein the mineral oxide comprisessilica.
 45. The chromatography column of claim 13, wherein the beads arecoated with a layer of a hydrophilic polymer.
 46. The chromatographycolumn of claim 13, wherein the apatite crystals comprise: (a) calciumions; and (b) a metal ion or a metalloid ion.